Our goal is to develop robust siRNA and micro-RNA methods to regulate genes for basic research, permitting both temporal and spatial control of transfection with high efficiency in both cell culture and C. elegans by using the unique chemical and physical properties of hollow gold nanoshells (HGN). HGNs are 30 - 40 nm diameter, 3-5 nm thick, gold shells designed to strongly absorb physiologically friendly NIR light and convert this light energy into local heating. HGN can be easily conjugated to small molecules, targeting ligands, polymers, siRNA and DNA by simple thiol chemistry, or incorporated into or tethered to liposomes. Water, proteins, lipids, etc. do not absorb NIR light, so cells in culture are essentially transparent, which eliminates damage during exposure. Femtosecond NIR light pulses trigger release of thiol-conjugated siRNA or other molecules from the HGN by breaking thiol bonds without damaging the siRNA. Even more important to the development of efficient oligonucleotide and small molecule delivery, the well-known bottleneck of endosomal escape can be bypassed by converting the physiologically friendly NIR light energy absorbed by the HGN to heat, creating unstable microbubbles that mechanically rupture endosomes and release siRNA to the cytosol within seconds. This allows much lower concentrations of HGN-siRNA conjugates to be used, greatly increases transfection efficiency, and provides spatially and temporally controlled transfection that can be used to pattern cultured cells and address specific structures within C. elegans. HGN transfection is as efficient as Lipofectamine, but is non-toxic, and can be used in living organisms. In this proposal, we will use the HGN-siRNA platform to develop masking and unmasking techniques for activating or inactivating biological processes with remote control to devise simple and scalable methods of lithographic patterning of cultured cells in real time. We will investigate controlled release of small molecules from liposomes incorporating HGN using NIR light triggering to control release, thereby enabling basic cell biological studies, including human stem cell differentiation, cancer biology, and signal transduction pathways and routes to applications in chemotherapy and drug delivery. We will develop methods to multiplex the release from a single HGN using a combination of thiol and dithiol anchors that desorbs at different energies to release of multiple chemical species. New silver nanoshells and gold and silver nanorods will be synthesized to probe other regions of the NIR spectrum and provide simultaneous delivery and imaging opportunities. These new constructs could be addressed independently by using different wavelength NIR irradiation. This project takes full advantage of the unique HGN interactions with physiologically friendly NIR light to initiate and control biological processes with precise spatial control at millisecond rates in living cells and organisms.